The ability to detect and/or quantitate the concentration of a pharmacological agent, metabolite, or toxin is a central aspect of the modem diagnosis and management of disease.
In some cases, such analytes can be detected directly, by assaying their biological activities. In most cases, however, it is more efficient to detect such molecules by virtue of their capacity to specifically bind to antibodies, or by their physical characteristics (such as electrophoretic mobility).
Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target analyte. The concept of immunoassays is based on a specific chemical reaction between an antibody and its corresponding antigen. Quantitation involves the separation of antibody bound antigen from the free antigen followed by detection of antibody bound antigen or free antigen in solution depending upon the specific analytical scheme. Such assays are used extensively in modem diagnostics (See, Fackrell, J. Clin. Immunoassay 8:213-219 (1985); Yolken, R. H., Rev. Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons, New York (1985); Ngo, T. T. et al., In: Enzyme Mediated Immunoassay, Plenum Press, New York (1985)).
There are many variations of immunoassay and the critical steps are either physical separation or discrimination and detection. Immunoassays that require physical separation are termed heterogeneous immunoassays. In contrast, homogeneous immunoassays are designed such that the removal of bound from unbound species is unnecessary. Because homogeneous assays lack a separation step, and are more easily automated, they are more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
Analytes present at concentration levels below 10.sup.-9 M are generally assayed using a solid-phase based "sandwich" or competitive method. Typically, in such assays, the antigen of interest competes with a labeled antigen for a judicious amount of antibody. A direct immunoassay is typically a sandwich assay involving two antibodies binding to different antigenic sites of an antigen. One antibody is bound to a solid phase material, and is employed to harvest the antigen. The other antibody is labeled and used to generate quantitative information from the bound antigen (Cone, E. J. et al., J. Forens. Sci. 35:786-781 (1990); Baugh, L. D. et al., J. Forens. Sci. 36:79-85 (1991); Standefer, J. C. et al., Clin. Chem. 37:733-738 (1991)).
In order to facilitate the detection of antibody binding, one or more reaction analytes is typically labeled (as with a radioisotope, an enzyme, a fluorescent moiety, a chemiluminescent moiety, or a macroscopic label, such as a bead, etc.) (see, Chard, T. et al., In: Laboratory Techniques and Biochemistry in Molecular Biology (Work, T. S., Ed.), North Holland Publishing Company, New York (1978); Kemeny, D. M. et al. (Eds.), ELISA and Other Solid Phase Immunoassays, John Wiley & Sons, New York (1988)). Radioisotopes have long been used in immunoassays. O'Leary, T. D. et al., for example describe a radioimmunoassay for digoxin serum concentrations (O'Leary, T. D. et al., Clin. Chem. 25:332-334 (1979)). The difficulty of handling such hazardous materials, and the problem of radioactive decay have led to the development of immunoassays that use other labels.
Enzymes, in particular, are now widely used as labels in immunoassay formats. The enzyme-multiplied immunoassay technique (EMIT.RTM., Syva Co.) has been used to assay acetaminophen, cocaine and other analytes (Helper, B. et al., Amer. J. Clin. Pathol. 81:602-610 (1984); Cambell, R. S. et al., J. Clin. Chem. Clin. Biochem. 24:155-159 (1986);Khanna, P., U.S. Pat. No. 5,103,021; Cone, E. J. et al., J. Forens. Sci. 35:786-781 (1990); Baugh, L. D. et al., J. Forens. Sci. 36:79-85 (1991); Standefer, J. C. et al., Clin. Chem. 37:733-738 (1991); Roberts, D. W. et al., J. Pharmacol. Exper. Therap. 241:527-533 (1987); Bartolone, J. B. et al., Biochem. Pharamcol. 37:4763-4774 (1988))
In addition to enzymes, fluorescent moieties are frequently used as labels (see, Ichinose, N. et al., In: Fluorometric Analysis in Biomedical Chemistry, Vol 110, Chemical Analysis (Winefordner, J. D. et al., Eds.) John Wiley & Sons, New York (1991)). For example, a fluorescence polarization immunoassay format for cocaine has been described (TDx.RTM., Abbott Laboratories, Inc.), and has been found to be approximately equivalent to the EMIT.RTM. formats (Schwartz, J. G. et al., Amer. J. Emerg. Med. 9:166-170 (1991)). The TDx.RTM. format has also been used to assay acetaminophen serum levels (Koizumi, F. et al., Tohoku J. Exper. Med. 155:159-(1988); Edinboro, L. E. et al., Clin. Toxicol. 29:241-(1991); Okurodudu, A. O. et al., Clin. Chem. 38:1040 (1992)), and serum digoxin levels (Okurodudu, A. O. et al., Clin. Chem. 38:1040 (1992)). Wong, S. H. Y. et al., have described the use of an automated (OPUS) analyzer to measure digoxin concentration in a monoclonal antibody mediated, fluorescence-based assay protocol (Wong, S. H. Y. et al., Clin. Chem. 38:996 (1992)). Lee, D. H. et al. also disclose the use of a fluorescence polarization assay and a chemiluminescent assay format to assay digoxin levels (Lee, D. H. et al., Clin. Chem. 36:1121 (1990)).
As indicated, electrophoretic methods have also been used to facilitate the detection of target analytes. Such methods exploit the fact that molecules in solution have an intrinsic electrical charge. Thus, in the presence of an electric field, each molecular species migrates with a characteristic "electrophoretic" mobility thereby causing the various species present to separate from one another. Under the influence of such a field, all of the variants will move toward a designated charge opposite to the charge of the variants; those having a lower electrophoretic mobility will move slower than, and hence be separated from, those having a (relative) higher electrophoretic mobility.
Immunological electrophoretic methods, such as Immunofixation electrophoresis ("IFE"), Immunoelectrophoresis ("IEP"), and immunosubtraction electrophoresis ("ISE") have been described which combine the capacity of electrophoretic methods to separate molecular species with the detection capacity of immunoassays. Such assays have been used to detect and quantitate serum proteins.
IEP and IFE are related procedures (Beckman Bulletin EP-2. "Immunoelectrophoresis Applications Guide." (1991)). IFE is a two stage procedure using agarose gel protein electrophoresis in the first stage and immunoprecipitation in the second. In a clinical setting for the analysis of immunoglobulins, a clinical sample (e.g., whole blood, serum, plasma, urine, cerebrospinal fluid) is placed in multiple positions ("lanes") on an agarose gel. When an electric field is applied to the gel-containing sample, the immunoglobulins will traverse the gel from anionic to cationic electrode. Thereafter, antisera comprising antibodies to specific immunoglobulin classes (typically IgG, IgA, IgM, kappa and lambda) are applied to specific lanes. The gel and antisera are incubated, during which time immune complexes form between the specific immunoglobulins and the antibodies. The location of such immune complexes are visualized by staining. By using a reference pattern on the gel, one can then determine the type of immunoglobulin present on the gel. The presence of a particular band is thus indicative of the presence of an M-protein corresponding to a particular immunoglobulin type. Methods of conducting IFE are disclosed by Chen, F-. T. A., U.S. Pat. No. 5,202,006; Chen, F-. T. A., U.S. Pat. No. 5,120,413; Hsieh, Y-. Z. et al., U.S. Pat. No. 5,145,567; all herein incorporated by reference).
The PARAGON.RTM. electrophoresis system (Beckman Instruments, Inc., Fullerton, Calif., U.S.A.) is a commercially available system for conducting both IFE and IEP (See also, Gebott et al., U.S. Pat. No. 4,669,363; Pentoney, S. L., U.S. Pat. No. 5,208,466, herein, herein incorporated by reference; Beckman Bulletin EP-3 "Paragon.RTM. Serum Protein Electrophoresis II (SPE-II) Applications Guide" (1990); Beckman Bulletin EP-2. "Immunoelectrophoresis Applications Guide" (1991); Beckman Bulletin EP-4 "Immunofixation Electrophoresis Applications Guide" (1991); Beckman Instructions 015-246513-H "Paragon.RTM. Electrophoresis System-IFE" (1990); Beckman Bulletin EP-6 "High Resolution Electrophoresis in the Detection of Monoclonal Gammopathies and Other Serum Protein Disorders." (1990); Chen, F-. T. A. et al. Clin. Chem. 37:14-19 (1991)).
Immunosubtraction electrophoresis (ISE) is a variation of IFE (Aguzzi, F. et al., Estratto dal. Boll. 1st Sieroter, Milanese 56:212-216 (1977); White, W. A. et al., Biochem. Clin. 10:571-574 (1986); Merlini, G. et al., J. Clin. Chem. Biochem. 21:841-844 (1983); Liu, C-. M. et al., U.S. Pat. No. 5,228,960, herein incorporated by reference). In ISE, however, the sample is pretreated with an insolubilized antibody directed to a particular "target" protein. If the target protein is present, it will bind to the antibody and thus be removed from the sample. The sample is then applied to a gel and subjected to electrophoresis. If the target protein had been present in the initial sample, visualization of the proteins in the gel would reveal a negative band (i.e. an absence of staining) at the position in the gel where the removed band would have migrated to, had it not been removed by the antibody. Thus, the absence of a particular band is indicative of the presence of the corresponding target protein in the sample.
IEP, IFE and ISE each require multiple steps, and the preparation and use of a separation gel and a signal-generating stain. The labor intensive nature of these procedure is an obvious impediment in a clinical setting. Additionally, the amount of disposable end-products associated with these procedures can further increase the allied costs associated with these procedures.
In view of the deficiencies of these methods in clinical settings, less labor-intensive methods that permit greater throughput with lower cost have been sought. One such method is "Capillary Electrophoresis" ("CE") (Chen, F-. T. A. et al., Clin. Chem. 77:14-19 (1991); Nielsen et al., J. Chromatogr. 539:177 (1991); U.S. Pat. No. 5,120,413, all herein incorporated by reference). Capillary electrophoresis (CE) is one of the most powerful tools yet developed for the separation of ionic species such as proteins, peptides and other water soluble molecules.
The method permits rapid and efficient separations of proteins (such as human growth hormone) (Grossman, P. et al., Anal. Chem. 61:1186-1194 (1989)), and other charged substances. Separation of the constituents of clinical samples can typically be accomplished in less than 20 minutes. Separation of proteins in plasma and serum sample have been attempted by Jorgenson, J. W. et al. (Science 222:266-272 (1983)) and Hjerten, S. (Electrophoresis 11:665-690 (1990)). The feasibility of routine analysis of serum proteins by CE in an untreated fused-silica capillary has been demonstrated (Chen, F-. T. A. et al., Clin. Chem. 37:14-19 (1991); Gordon, M. G. et al., Anal. Chem. 63:69-72 (1991)).
In general, CE involves introducing a sample into a capillary tube, i.e. a tube having an internal diameter of from about 2 .mu.m to about 2000 .mu.m (preferably, less than about 50 .mu.m, most preferably, about 25 .mu.m or less) and applying an electric field to the tube (Chen, F-. T. A., J. Chromatogr. 516:69-78 (1991); Chen, F-. T. A. et al., J. Chromatogr. 15:1143-1161 (1992)). Since each of the sample constituents has its own individual electrophoretic mobility, those having greater mobility travel through the capillary tube faster than those with slower mobility. Hence, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. (Heegard, N. H. H. et al., Anal. Chem. 64:2479-2482 (1992); Gordon, M. J. et al., Anal. Chem. 63:69-72 (1991); F-. T. A., U.S. Pat. No. 5,202,006; Chen, F-. T. A., U.S. Pat. No. 5,120,413; Hsieh, Y-. Z. et at., U.S. Pat. No. 5,145,567). The method is well-suited to automation, since it provides convenient on-line injection, detection and real-time data analysis. Detection of protein in CE is usually based on the intrinsic ultraviolet (UV) absorbance of the peptide bond at or near 200 nm, which provides a detection limit of about 10.sup.-5 M. Fluorescence-based detection assays have, however, also been described (Lee, T. T. et al., J. Chromatogr. 595:319-325 (1992)).
The capillary column used in CE must be capable of withstanding a wide range of applied electrophoretic fields of between about 10 v/cm to about 1000 v/cm. Fused silica is a preferred material for the capillary tube because it can withstand such voltages, and because the inner walls ionize to create the negative charge which causes the desired electroosmatic flow.
In some cases, however, the use of fused silica can have undesired effects. Proteins, for example, are polyelectrolytes, consisting of both positively and negatively charged moieties. The pKa of the guanidinium and e-NH.sub.2 groups of the arginine and lysine residues, respectively, is 12.0 and 10.5, and these groups comprise most of the positively charged moieties in proteins, other than the a-NH2 terminal (which has a pKa of between 7.5 and 9) and histidine residues. Because a fused-silica surface contains weakly acidic silanol groups that act as cation-exchangers, the protein-silica surface interaction can be viewed as an ion-exchange phenomenon (Lauer, H. H. et al., Anal. Chem. 58:166-169 (1986); Green, J. S. et al., J. Chromatogr. 478:63-70 (1989)). Early efforts to electrophorese proteins in untreated fused-silica capillaries resulted in broad peaks and irreproducible migration of the sample zones (Jorgenson, J. W. et al., Science 222:266-272 (1983)). The interaction of proteins with the silica wall is believed to be responsible for degrading the efficiency of protein separations by CE in untreated fused-silica capillaries.
Capillaries that have been coated with a material such as alumina, beryllium, Teflon.RTM., glass, quartz, etc. or combinations thereof, have been found to limit protein absorption to the untreated walls during the electrophoretic separation procedure. Such coated capillaries have enjoyed widespread use. However, eventually these coatings break down in an unpredictable manner. Thus, the use of uncoated capillaries has been generally preferred.
To avoid or minimize the protein-silica interaction, protein separations by CE in untreated fused-silica capillaries have previously been performed in buffers having a pH either substantially higher than the isoelectric points of the sample proteins (Lauer, H. H. et al., Anal. Chem. 58:166-169 (1986)), or .ltoreq.2.5 (McCormick, R., Anal. Chem. 60:2322-2327 (1988)), or in a relatively basic buffer with a high salt concentration (Greene, J. S. et al., J. Chromatogr. 478:63-70 (1989)). Greene, J. S. et al. (J. Chromatogr. 478:63-70 (1989)) demonstrated the high efficiency of separation of model proteins by CE in untreated fused-silica column using 0.1M CHES (CHES: 2-(Cyclohexylamino)ethanesulfonic acid) buffer in 0.25M potassium sulfate at pH 9.0. However, using a similar zwitterionic buffer system such as BES (BES: N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) and HEPES (HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid;) at pH 7.0 and 8.0, respectively, in 0.25M potassium sulfate, model proteins are poorly resolved. As the pH of the buffer decreases from 9.0 to 7.0, proteins become more positively charged, thus increasing the protein-silica wall interaction. To avoid this increased interaction, the amount of potassium sulfate has to be increased, resulting in a higher conductivity in CE. Furthermore, proteins in complex mixtures, such as serum, ascites fluid and tissue extracts, are relatively difficult to separate, due to the presence of lipids and their associated proteins.
In view of the importance of accurately detecting and quantitating analyte, and especially protein, concentrations in samples, it would be particularly desirable to possess a generally applicable methods would allow for separation of model proteins and complex protein mixtures. A capillary electrophoresis technique that could additionally be employed to resolve organic analytes (such as pollutants, toxins, etc.) and which could provide a facile means of detection would also be highly desirable. The present invention provides such methods.